High expression FUT1 and B3GALT5 is an independent predictor of postoperative recurrence and survival in hepatocellular carcinoma.

Cancer may arise from dedifferentiation of mature cells or maturation-arrested stem cells. Previously we reported that definitive endoderm from which liver was derived, expressed Globo H, SSEA-3 and SSEA-4. In this study, we examined the expression of their biosynthetic enzymes, FUT1, FUT2, B3GALT5 and ST3GAL2, in 135 hepatocellular carcinoma (HCC) tissues by qRT-PCR. High expression of either FUT1 or B3GALT5 was significantly associated with advanced stages and poor outcome. Kaplan Meier survival analysis showed significantly shorter relapse-free survival (RFS) for those with high expression of either FUT1 or B3GALT5 (P = 0.024 and 0.001, respectively) and shorter overall survival (OS) for those with high expression of B3GALT5 (P = 0.017). Combination of FUT1 and B3GALT5 revealed that high expression of both genes had poorer RFS and OS than the others (P < 0.001). Moreover, multivariable Cox regression analysis identified the combination of B3GALT5 and FUT1 as an independent predictor for RFS (HR: 2.370, 95% CI: 1.505-3.731, P < 0.001) and OS (HR: 2.153, 95% CI: 1.188-3.902, P = 0.012) in HCC. In addition, the presence of Globo H, SSEA-3 and SSEA-4 in some HCC tissues and their absence in normal liver was established by immunohistochemistry staining and mass spectrometric analysis

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Chimeras of p14ARF and p16: functional hybrids with the ability to arrest growth.

The INK4A locus codes for two independent tumor suppressors, p14ARF and p16/CDKN2A, and is frequently mutated in many cancers. Here we report a novel deletion/substitution from CC to T in the shared exon 2 of p14ARF/p16 in a melanoma cell line. This mutation aligns the reading frames of p14ARF and p16 mid-transcript, producing one protein which is half p14ARF and half p16, chimera ARF (chARF), and another which is half p16 and half non-p14ARF/non-p16 amino acids, p16-Alternate Carboxyl Terminal (p16-ACT). In an effort to understand the cellular impact of this novel mutation and others like it, we expressed the two protein products in a tumor cell line and analyzed common p14ARF and p16 pathways, including the p53/p21 and CDK4/cyclin D1 pathways, as well as the influence of the two proteins on growth and the cell cycle. We report that chARF mimicked wild-type p14ARF by inducing the p53/p21 pathway, inhibiting cell growth through G2/M arrest and maintaining a certain percentage of cells in G1 during nocodazole-induced G2 arrest. chARF also demonstrated p16 activity by binding CDK4. However, rather than preventing cyclin D1 from binding CDK4, chARF stabilized this interaction through p21 which bound CDK4. p16-ACT had no p16-related function as it was unable to inhibit cyclin D1/CDK4 complex formation and was unable to arrest the cell cycle, though it did inhibit colony formation. We conclude that these novel chimeric proteins, which are very similar to predicted p16/p14ARF chimeric proteins found in other primary cancers, result in maintained p14ARF-p53-p21 signaling while p16-dependent CDK4 inhibition is lost

Concordant and Discordant Regulation of Target Genes by miR-31 and Its Isoforms

<div><p>It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA) variants designated as “isomiR”. Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M), which differ only slightly in their 5′- and/or 3′-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3′-untranslated region (UTR) of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.</p> </div

Concordant and discordant regulation of known target genes by isomiR-31s.

<p>The sequences of isomiRs of miR-31. MiR-31-H, miR-31-P, and miR-31-M represent hsa-miR-31, ptr-miR-31, and mmu-miR-31 in miRBase, respectively (A). CEBPα, STK40, and E2F2 mRNA expression in MDA-MB-231 cells (B), MCF-7 cells (C), and in HCT116 cells (D) were detected by RT-qPCR after transfection with synthetic oligos of isomiR-31s. The mRNA level of each gene was normalized to GAPDH mRNA. The normalized mRNA level of Neg-ctrl transfectant was set as 1.0 and then those of other isomiR-31 transfections were relative to it. The proteins levels of Fzd3 (E), MMP16 (F), and MCM2 (G) were determined in MDA-MB-231 cells transfected with 100 nM synthetic oligos by immunoblotting. GAPDH protein served as the internal control for normalization. The normalized protein level of Neg-ctrl transfectant was set as 1.0 for comparison to those of isomiR-31 transfectants. The data represent the average of 3 independent experiments with standard deviations (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001, t-test).</p

MiR-31-P enhanced the sensitivity of cancer cells to cisplatin treatment.

<p>IsomiR-31 transfected MCF-7 breast cancer cells (A) and A549 lung cancer cells (B) were incubated with cisplatin at the indicated concentrations. At 48 h, the numbers of surviving cells were analyzed by Alamar Blue reagent and the percentages of cell survival were listed. The percentage of surviving cells of each transfected groups treated with DMSO was set as 100% to calculate the percentages of surviving cells of cisplatin treated cells at the indicated concentration. Comparing to the negative control transfected cells, miR-31-P enhanced the sensitivity of both cancer cells to cisplatin treatment (*<i>P</i><0.05; **<i>P</i><0.01, t-test). The statistical significance of the differential sensitivity to cisplatin of MCF-7 (C) and A549 (D) cells transfected with various isomiR-31s was further examined by nonlinear regression analysis (GraphPad Prism software version 5.01). Nonlinear regression analysis was used to provide the best fitted sigmoid curves by plotting the percentages of cell survival against the drug concentrations (*<i>P</i><0.05; **<i>P</i><0.01, ANOVA). The data represent the average of 3 independent experiments with standard deviations.</p

The most abundant isoform and the composition of miR-31 populations vary among five human cells.

<p>IsomiR-31s in MCF-7, HCT116, and LNCaP cells was analyzed by deep sequencing and compared to the reported miR-31 isoforms in human embryonic stem cell (hES)/embryonic body (hEB) culled from the supplementary data of Morin <i>et.al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058169#pone.0058169-Morin1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058169#pone.0058169-Voellenkle1" target="_blank">[32]</a>. The miR-31 precursor sequence is shown at the bottom. The sequences, which is underlined with thick line or marked with <b>^*</b>, is the current annotated miR-31 of human in miRBase (version 18.0). The occurrence of each sequence read is represented as the count shown in number. The percentage of each sequence indicates its occurrence in the whole population of miR-31 isoforms. In the miR-31 profile of HCT116 cells, most of sequences with counts of less than 10 were omitted from this figure. ^#, the data were culled from the report of Morin <i>et al</i>. H, hsa-miR-31; the miR-31-H form. M, mmu-miR-31; the miR-31-M form. P, ptr-miR-31; the miR-31-P form.</p